CN106676087B - A kind of preparation method of proline restriction endonuclease - Google Patents
A kind of preparation method of proline restriction endonuclease Download PDFInfo
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- CN106676087B CN106676087B CN201710069338.6A CN201710069338A CN106676087B CN 106676087 B CN106676087 B CN 106676087B CN 201710069338 A CN201710069338 A CN 201710069338A CN 106676087 B CN106676087 B CN 106676087B
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- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21026—Prolyl oligopeptidase (3.4.21.26), i.e. proline-specific endopeptidase
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Abstract
The invention discloses a kind of preparation method of proline restriction endonuclease, including the preparation of aspergillus niger kind liquid, cultivation and fermentation, broth extraction processing：It is BMGY fluid nutrient mediums that kind liquid, which prepares used medium, and condition of culture is 28 35 DEG C, 200 250r/min Shaking cultures, and incubation time is 12 18h；By obtained kind of liquid Shaking culture, culture medium is aspergillus niger high density fermentation culture medium, and condition of culture is 28 35 DEG C, 200 250r/min, or by obtained kind of liquid fermentation tank culture, culture medium is aspergillus niger high density fermentation culture medium, condition of culture is 28 35 DEG C, 200 800r/min；Zymotic fluid is filtered, concentration, after allotment, refined filtration or is dried to obtain liquid proline restriction endonuclease or solid proline restriction endonuclease.The present invention uses natural aspergillus niger strain, and using proline as inducing substrate, the proline restriction endonuclease that enzyme activity is high, temperature and pH accommodations are wide, has good stability is prepared under gentle fermentation condition.
Description
Technical field
The present invention relates to microbial fermentation engineering, more particularly, to a kind of preparation method of enzyme preparation, specifically a kind of dried meat
The preparation method of propylhomoserin restriction endonuclease.
Background technology
Aspergillus niger (Aspergillus niger) source proline endo protease (prolyl endoprotease,
PEP) be it is a kind of can be with effectively hydrolyzing protein or the protease of polypeptide proline residue carboxy-terminal peptide bond.PEP can extensive use
Muddy, tea cream forms and reduced protolysate bitter taste after beer is prevented.Domestic market is also without the PEP of independent research
Enzyme preparation product, it is completely dependent on import.Filamentous fungi has powerful secreting, expressing ability, and it is homologous or different to be widely used in expression
Source protein.Aspergillus niger is modified after having complete and efficient protein translation as basic security (GRAS) microorganism, same in expression
When source or heterologous protein, have safety, high yield and it is efficient the characteristics of.
In Food enzyme industry, usually requiring that the enzyme of industrial applications has higher stability and suitable optimal pH
Value and relatively low production cost.The proline Inner peptases of Aspergillus niger origin can act on the larger albumen of molecular weight (35kDa)
And polypeptide, the substrate spectrum of effect are wide.The heat endurance of the proline endo protease of Aspergillus niger origin is good simultaneously, in pH5.0,
30min is incubated under the conditions of 65 DEG C, does not detect the reduction of proline endoproteolytic enzyme activity.
The method that proline endo protease mainly takes genetic engineering, example are produced currently with aspergillus especially aspergillus niger
As Chinese patent CN200710150282.3, CN201610202929.1, CN201310656778.3 all refer to produce proline
The method of endo protease, used technological means are to obtain target enzyme system by genetic recombination mode culturing engineering bacterium
Agent, process is complicated, it is necessary to determine the amino acid sequence of target enzyme preparation, designs relevant primer, is unfavorable for industrial promoting production.
The present invention is adopted using aspergillus niger as production bacterium, and the dried meat in aspergillus niger is directly induced by induced expression substrate of proline
The high efficient expression of propylhomoserin incision enzyme gene.
The content of the invention
In order to solve the above technical problems, the present invention provides a kind of preparation method of new proline restriction endonuclease, by highly dense
Degree fermentation adds appropriate induced expression substrate proline to induce the expression of proline incision enzyme gene in aspergillus niger, so as to
To proline restriction endonuclease, the enzyme preparation can be widely applied to field of food.
In order to achieve the above object, the technical scheme is that providing a kind of preparation method of proline restriction endonuclease, bag
The preparation of aspergillus niger kind liquid, cultivation and fermentation, broth extraction processing are included, step is specific as follows：
(1) preparation of liquid is planted：By aspergillus niger strain after culture medium flat plate activation, strain is taken to access BMGY fluid nutrient mediums
In, 28-35 DEG C, 200-250r/min Shaking cultures to thalline OD600nm be 2-3, cultivate 12-18h, obtain kind of a liquid；
(2) cultivation and fermentation：Thalline is collected by centrifugation in obtained kind of liquid, and with after aseptic distillation water washing, accesses aspergillus niger
In high density fermentation culture medium, 28-35 DEG C, the fermentation of 200-250r/min Shaking cultures, proline is added every 16-48h, to dried meat
The enzyme activity of propylhomoserin restriction endonuclease is slowly raised or no longer raised, and terminates fermentation, fermentation duration 96-200h；The dense 5-20g/L of zymophyte
(in terms of thalline weight in wet base)；
Or liquid will be planted made from step (1), access in aspergillus niger high density fermentation culture medium, 28-35 DEG C, 200-
800r/min fermentation tank cultures are fermented, and the flow velocity of proline are added according to the adjustment of the state of fermentation tank pH and dissolved oxygen, to proline
The enzyme activity of restriction endonuclease is slowly raised or no longer raised, and terminates fermentation, fermentation duration 96-200h；The dense 100-300g/L of zymophyte
(in terms of thalline weight in wet base)；
(3) zymotic fluid is filtered, concentration, after allotment, refined filtration or is dried to obtain liquid proline restriction endonuclease or solid dried meat
Propylhomoserin restriction endonuclease.
As a further improvement, the BMGY fluid nutrient mediums percentage by weight composition in the step (1) is：Tryptone
20g/L, yeast extract 10g/L, 121 DEG C, after the 20min that sterilizes, add 100mmol/L pH6 potassium phosphate, no amino nitrogen source
13.4g/L, biotin 4 × 10-4G/L, glycerine 10g/L.
As a further improvement, the aspergillus niger high density fermentation culture medium in the step (2) is by following weight percentage
Component is prepared：Sucrose 5-15%, corn flour 5-6%, beancake powder 1.5-2.5%, glucose 3-4%, dusty yeast 0.4-
0.8%, corn steep liquor 0.1-0.5%, ammonium sulfate 0.05-0.4%, dipotassium hydrogen phosphate 0.05-0.4%, potassium dihydrogen phosphate 0.05-
0.4%, sodium citrate 0.1-0.5%, defoamer 0.1-1mL, pH value 5.0-7.0,121 DEG C of sterilizing 20min.Prepared using special
Fermentation tank culture medium, while cell concentration when controlling fermentation termination can ensure further to improve the enzyme of proline restriction endonuclease
It is living.
As a further improvement, it is 5000- that parameter that thalline uses, which is collected by centrifugation, as centrifugation rate in the step (2)
10000r/min, centrifuge 5-10min.
As a further improvement, the concentration of proline added in the step (2) is 0.1-0.5mg/L.With suitable concn
Proline be inducing substrate, and by feed profile addition can effectively induce high enzyme activity proline restriction endonuclease generation.
As a further improvement, pH maintains 5.0-7.0 in fermentation tank in the step (2), dissolved oxygen maintains 35-
50%.
Compared with prior art, the present invention uses natural aspergillus niger strain, safe, using proline as induction bottom
Thing, the culture medium of certain component is coordinated in shaking flask or fermentation tank, and can to obtain zymologic property under gentle fermentation condition good
Good proline restriction endonuclease；The condition of fermented and cultured is easily controlled, and method is simple and easy to operate；Proline prepared by the inventive method
Its temperature of restriction endonuclease and pH accommodations are wide, have good stability, and below 10 DEG C Cord blood after 12 months enzyme activity loss it is small
In 10%, zymotic fluid enzyme activity is up to 0.1-10PPU/mL.
Brief description of the drawings
Fig. 1 is the relative activity curve of proline restriction endonuclease under different pH；
Fig. 2 is the relative activity curve of proline restriction endonuclease under different temperatures.
Embodiment
With reference to embodiment, the invention will be further described, unless stated otherwise, technology hand used in the present invention
Duan Junwei methods known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Scope, the spirit and scope of the invention are limited only by the claims that follow.Unless otherwise instructed, agents useful for same is equal in embodiment
To meet the commercially available prod of fermentation process.
The aspergillus niger Aspergillus niger of embodiment 1 shaking flask high density fermentation
Step 1：The aspergillus niger glycerine strain of conventional -80 DEG C of preservations of glycerine is lined on malt extract medium flat board, put
Put in 30 DEG C of incubators and cultivate 2 days, until growing single bacterium colony；The full single bacterium colony streak inoculation of picking is in malt extract medium again
On flat board, place after being cultivated 2 days in 28 DEG C of incubators, take full strain to access in 50mLBMGY fluid nutrient mediums, 28 DEG C,
200rpm Shaking culture 18h, obtain kind of a liquid.
Wherein BMGY fluid nutrient mediums percentage by weight, which forms, is：Tryptone 20g/L, yeast extract 10g/L, 121 DEG C,
After sterilizing 20min, 100mmol/L pH 6 potassium phosphate, no amino nitrogen source 13.4g/L, biotin 4 × 10 are added-4G/L (filterings
Sterilizing), glycerine 10g/L.
Step 2：In superclean bench gnotobasis, obtained kind of liquid is centrifuged into 10000r/ under the protection of flame
Min, 10min, thalline is collected, and after being shaken with sterile purified water and washing twice, access the aspergillus niger high density fermentation training being prepared
Support in base, 28 DEG C, the fermentation of 200rpm/min Shaking cultures；0.5mg/L proline is added every 24h, to proline restriction endonuclease
Enzyme activity slowly rise or no longer raise, after fermented and cultured 120h, terminate fermentation, the dense 5-20g/L of zymophyte is (with thalline weight in wet base
Meter).
Wherein aspergillus niger high density fermentation culture medium, which forms, is：Sucrose 5-15%, corn flour 5-6%, beancake powder 1.5-
2.5%, glucose 3-4%, dusty yeast 0.4-0.8%, corn steep liquor 0.1-0.5%, ammonium sulfate 0.05-0.4%, dipotassium hydrogen phosphate
0.05-0.4%, potassium dihydrogen phosphate 0.05-0.4%, sodium citrate 0.1-0.5%, defoamer 0.1-1mL, pH value 5.0-7.0,
121 DEG C of sterilizing 20min.
The aspergillus niger Aspergillus niger of embodiment 2 fermentation tank high density fermentation
Step 1：With embodiment 1；
Step 2：In sterile room, by obtained kind of liquid, aspergillus niger high density fermentation culture medium (same embodiment is accessed
1) in, fermentation medium pH is adjusted to 5.8,30 DEG C, 200-800r/min cultivation and fermentations；According to fermentation tank pH and dissolved oxygen state
The flow velocity of 0.5mg/L proline, pH maintenance 5.0-7.0, between dissolved oxygen maintains 35-50%, to proline inscribe are added in adjustment
The enzyme activity of enzyme is slowly raised or no longer raised, and terminates fermentation, and ferment duration 96-200h, and the dense 100-300g/L of zymophyte is (with bacterium
Body weight in wet base meter).
The optimal pH and stability of the proline restriction endonuclease of embodiment 3
Using proline as substrate, the optimal pH and stability of proline restriction endonuclease are determined.Enumerate table 1 shown it is each
Under the conditions of kind pH, relative activity is recorded after being incubated 30 minutes at 37 DEG C, draws out the pH curves shown in Fig. 1.According to the curve
Understand, enzyme shows most highly actives 5.0 times in pH, and relative activity is more than 60% between pH 3.5-8.0, it may be determined that it is most suitable
PH scopes are 3.5-8.0, and are 4.5-5.5 relative to the optimal pH of the natural proline restriction endonuclease of aspergillus niger, side of the invention
Method substantially expands the optimal pH scope of proline restriction endonuclease.Simultaneously from the pH stability of enzyme, pH3.0-7.0 it
Between, it remains more than the 80% of initial activity.
Table 1
It was found from above-mentioned data, proline restriction endonuclease of the invention can keep stability in the range of larger pH, and
Suitable for playing enzymatic activity within the range.
The optimum temperature and stability of the proline restriction endonuclease of embodiment 4
Using proline as substrate, the optimum temperature and stability of prolyl restriction endonuclease are determined.Enumerate what table 2 was shown
Under various temperature conditionss, relative activity is recorded after being incubated 30 minutes when pH is 5, draws out the temperature curve shown in Fig. 2.Root
According to temperature curve, enzyme shows most highly active at 45 DEG C, in 25 DEG C of -60 DEG C of relative activities more than 60%, relative to from black song
The optimum temperature of mould natural proline restriction endonuclease is 35 DEG C -45 DEG C.Simultaneously from the temperature stability of enzyme, 25 DEG C-
50 DEG C, it remains more than the 80% of initial activity.
Table 2
The Enzyme activity assay method of the proline restriction endonuclease of embodiment 5
Principle：Specific proline restriction endonuclease belongs to serine stretch protein enzyme family, can be residual to the proline inside polypeptide
Base carries out specific cutting.This method is with carbon teminal benzyl protection, and proline of the nitrogen end using paranitroanilinum as protection group of is at the bottom
Thing, enzyme activity calculating is carried out by detecting the paranitroanilinum concentration in enzymatic system；Enzyme-activity unit is defined as every milliliter of enzyme liquid
Interior generation 1umol paranitroanilinum per minute is an enzyme-activity unit.
Enzyme activity determination preparation of reagents：
Mother liquor A (250 μM of Z-Gly-Pro-pNA (M=426.43))：It is molten accurately to weigh 0.0053g Z-Gly-Pro-pNA
Solution is after 50mL Isosorbide-5-Nitraes-dioxane (volume ratio 40%), fully ultrasonic wave hydrotropy, dissolving, the packing of 2mL centrifuge tubes, and preserves
In -20 DEG C.
Mother liquor B (200mM potassium phosphates (M=212.27) solution)：It is fixed after accurately weighing the steaming water dissolving of 42.45g potassium phosphates list
Hold to 1L；
Mother solution C (200mM acetic acid (M=60.05) solution)：Accurate to weigh 12.01g acetic acid, single water that steams is settled to 1L；
Working solution D (acetic acid/kaliumphosphate buffer (100mM, pH5.0))：Mother liquor B, C are diluted 2 times with single water that steams respectively
Afterwards, it is mutually mixed under pH monitorings, is configured to pH5.0,100mM working solution；
Mother liquor E (paranitroanilinum (M=138.13), 50mM, is prepared)：Accurately weigh 0.173g paranitroanilinum, recruitment
Make liquid D dissolvings, constant volume to 50mL；
The drafting of paranitroanilinum standard curve：Mother liquor E is drawn in test tube, final concentration of 5 are diluted to working solution D,
10,20,40,75,100,150 μM, cumulative volume treats test sample for 10mL's, fully mixes, numbering 1,2,3,4,5,6,7,8；Point
After light photometer preheating 20min, returned to zero at 410nm with working solution D；Each sample repeats to take 2.5mL respectively after mixing,
The absorption value of testing sample 1-8 is determined under the conditions of 410nm, each sample replication 3 times, records experimental data；To being remembered
The experimental data of record calculates average value, and using absorption value as X-axis, concentration is Y-axis, draws standard curve, adds Trendline, foundation pair
The basic calculating formula of nitroaniline absworption peak and concentration；
Y=а X
Enzyme activity determination step：
Zymotic fluid 1mL, 10000rpm/min centrifugation 2min is taken, takes 4 DEG C of supernatant to save backup；Take working solution D 1025mL
In quartz colorimetric utensil, and add mother liquor A 475uL and returned to zero at 410nm；1000uL PESP fermentation supernatants enzyme liquid is taken to add ratio
In color ware, rapid inhale is beaten uniformly；Light absorption value is determined at 410nm, timing 10min, a data are recorded every 60s；Repeat to survey
Determine three times；Calculate the difference DELTA Abs of absorption value in 10 minutes
Enzyme activity calculates：
Enzyme activity (PESPU) calculation formula is PESPU/mL=Δ Abs* а * E/ (2.5*0.025) (PPU/mL)
ΔAbs:At 410nm in absworption peak 10 minutes absorption value difference；а：Criterion calculation constant；E：Fermented supernatant fluid is dilute
Release multiple；
Enzyme activity determination result is as shown in table 3
Table 3
The present embodiments relate to material, reagent and experimental facilities, unless otherwise instructed, be meet microorganism hair
The commercially available prod in ferment enzyme field.
It is described above, only the preferred embodiments of the present invention, it is noted that for those skilled in the art
For, on the premise of the core technology of the present invention is not departed from, improvements and modifications can also be made, these improvements and modifications also should
Belong to the scope of patent protection of the present invention.Any change in the implication and scope suitable with claims of the present invention, all
It is considered as being included within the scope of the claims.
Claims (7)
- A kind of 1. preparation method of proline restriction endonuclease, it is characterised in that step include aspergillus niger kind liquid prepare, using proline as The fermentation of induced expression substrate cultivation, broth extraction processing；Prepared by described kind of liquid concretely comprises the following steps：By aspergillus niger strain in culture After base activation, take in strain access BMGY fluid nutrient mediums, 28-35 DEG C, 200-250r/min Shaking culture 12-18h, to bacterium Body OD600nm is 2-3, obtains kind of a liquid；The cultivation and fermentation first method step is：Thalline, access aspergillus niger high density hair is collected by centrifugation in obtained kind of liquid In ferment culture medium, 28-35 DEG C, the fermentation of 200-250r/min Shaking cultures, it is 0.1-0.5mg/L's to add concentration every 16-48h Proline, fermentation duration 96-200h；Or the cultivation and fermentation second method step is：By obtained kind of liquid, aspergillus niger high density fermentation culture medium is accessed In, 28-35 DEG C, the fermentation of 200-800r/min fermentation tank cultures, proline is added according to the adjustment of the state of fermentation tank pH and dissolved oxygen Flow velocity, fermentation duration 96-200h.
- 2. the preparation method of proline restriction endonuclease according to claim 1, it is characterised in that the broth extraction processing Step is：Zymotic fluid is filtered, concentration, after allotment, refined filtration or is dried to obtain liquid proline restriction endonuclease or solid proline Restriction endonuclease.
- 3. the preparation method of proline restriction endonuclease according to claim 1, it is characterised in that the aspergillus niger high density hair Ferment culture medium is made up of following weight percent composition：Sucrose 5-15%, corn flour 5-6%, beancake powder 1.5-2.5%, glucose 3-4%, dusty yeast 0.4-0.8%, corn steep liquor 0.1-0.5%, ammonium sulfate 0.05-0.4%, dipotassium hydrogen phosphate 0.05-0.4%, di(2-ethylhexyl)phosphate Hydrogen potassium 0.05-0.4%, sodium citrate 0.1-0.5%, defoamer 0.1-1mL, pH value 5.0-7.0,121 DEG C of sterilizing 20min.
- 4. the preparation method of proline restriction endonuclease according to claim 1, it is characterised in that the cultivation and fermentation the first It is 5000-10000r/min that parameter that thalline uses is collected by centrifugation described in method and step as centrifugation rate, centrifuges 5-10min.
- 5. the preparation method of proline restriction endonuclease according to claim 1, it is characterised in that the cultivation and fermentation the first When the fermentation of Shaking culture described in method and step terminates, strain concentration is counted as 5-20g/L using thalline weight in wet base.
- 6. the preparation method of proline restriction endonuclease according to claim 1, it is characterised in that second of the cultivation and fermentation PH described in method and step maintains 5.0-7.0, and dissolved oxygen maintains 35-50%.
- 7. the preparation method of proline restriction endonuclease according to claim 1, it is characterised in that second of the cultivation and fermentation When the fermentation of fermentation tank culture described in method and step terminates, strain concentration is counted as 100-300g/L using thalline weight in wet base.
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